The long-term objectives of this project are to elucidate: (i) the developmental regulation of the beta-globin gene family, and (ii) the role of the nuclear factor GF-1 in the control of globin genes have long been studied as a model system for gene regulation, and with a view to the eventual treatment of hemoglobinopathies. Recently, the use of transgenic mice has been instrumental in defining cis-acting elements involved in temporal and tissue-specific control of the gene family. Continuing our studies of beta-globin regulation, we will employ two new strategies to manipulate regulatory elements without disrupting the overall organization of the locus, which appears to be functionally important. First, the use of yeast artificial chromosomes will make possible the cloning and manipulation of the entire human gene cluster, and its subsequent transfer into the mouse germ line, either in wild type form or after introducing specific mutations. Second, gene targeting techniques will permit the introduction of site-directed mutations into the endogenous beta-globin locus in murine embryonic stem (ES) cells. As well as testing models of regulation, the latter experiments should result in the production of new murine models of beta-thalassemia. GF-1 is a DNA-binding protein found specifically in erythroid, megakaryocyte and mast cells, which appears to play a major role in the control of cell type-specific genes, and thus in cell differentiation. We propose to investigate the role of this nuclear factor in the process of hemopoietic cell differentiation, and particularly in globin gene expression, via several approaches: first, we will study the developmental consequences of a mutation that has disrupted the X- linked GF-1 gene in a male ES cell line, by examining the ability of the mutant cells to populate hemopoietic lineages in chimeric mice, and by attempting to pass the mutation through the germ line; second, we will use transgenic techniques to alter the level and pattern of GF-1 expression in the developing mouse; and finally, we will perform experiments to define the regulatory elements involved in the activation of the GF-1 gene itself, which may represent a key step in the commitment of hemopoietic progenitors to specific changes.